The present invention relates to an isolated (trans,cis)-10,12-linoleate isomerase enzyme, to a nucleic acid molecule encoding a (trans,cis)-10,12-linoleate isomerase enzyme, to immobilized cells containing a linoleate isomerase enzyme, to an immobilized (trans,cis)-10,12-linoleate isomerase enzyme, and to a method for converting linoleic acid or linolenic acid to CLA or derivatives thereof using the isolated linoleate isomerase enzyme, nucleic acid molecule and/or immobilized cells.
The term xe2x80x9cCLAxe2x80x9d is used herein as a generic term to describe both conjugated linoleic acid and conjugated linolenic acid. The CLA compounds (cis,trans)-9,11-linoleic acid and (trans,cis)-10,12-linoleic acid are recognized nutritional supplements and effective inhibitors of epidermal carcinogenesis and forestomach neoplasia in mice, and of carcinogen-induced rat mammary tumors. CLA has also been shown to prevent adverse effects caused by immune stimulation in chicks, mice and rats, and has been shown to decrease the ratio of low density lipoprotein cholesterol to high density lipoprotein cholesterol in rabbits fed an atherogenic diet. CLA also reduces body fat in mouse, rat, chick and pig models. CLA has also been shown to be effective in treating skin lesions when included in the diet.
CLA occurs naturally in various amounts in virtually all foods. The principle natural sources of CLA are dairy products, beef and foods derived from ruminant animals. In the U.S., beef, beef tallow, veal, lamb (3-4 mg CLA/g fat; 84% cis-9,trans-11) and dairy products (3-7 mg CLA/g fat; 80-90% cis-9,trans-11) have the highest concentration of CLA. CLA concentrations 2-3 times higher are found in Australian dairy products and pasture-fed beef and lamb. Very low concentrations of CLA (0.1-0.7 mg CLA/g fat; ca. 40% each cis-9,trans-11 and trans-10,cis-12) are found in commercial vegetable oils.
CLA is a normal intermediate of linoleic acid metabolism. In cows, (cis,trans)-9,11-CLA produced by natural bacterial flora that is not further metabolized is incorporated into lipids and then into host tissues and milk. Animals take up and incorporate CLA into normal tissue and milk from dietary sources such as milk, milk products or meat containing CLA, or from CLA dietary supplements.
CLA can be synthetically obtained from alkaline isomerization of linoleic or linolenic acid, or of vegetable oils which contain linoleic acid, linolenic acid or their derivatives. Heating vegetable oil at about 180xc2x0 C. under alkaline conditions catalyzes two reactions: (1) fatty acid ester bonds from the triglyceride lipid backbone are hydrolyzed, producing free fatty acids; and (2) unconjugated unsaturated fatty acids with two or more appropriate double bonds are conjugated. Commercial CLA oils available at the present time, typically made from sunflower oil, are sold without further purification. They contain a mixture of CLA isomers as well as other saturated and unsaturated fatty acids. Generally, chemical synthesis produces about 20-35% (cis,trans)-9,11-CLA and about 20-35% (trans,cis)-10,12-CLA, and the balance as a variety of other isomers. The presence of the non-active, non-natural isomers introduces the need to purify (cis,trans)-9,11-CLA and/or (trans,cis)-10,12-CLA, or to demonstrate the safety and seek regulatory approval of these non-beneficial, non-natural isomers for human use. It is not feasible economically, however, to isolate single isomers of CLA from the CLA made by alkaline isomerization. Using a fractional crystallization procedure, it is possible to enrich 9,11-CLA relative to 10,12-CLA and vice versa. U.S. Pat. No. 6,015,833, issued Jan. 18, 2000, to Saebxc3x8 et al. describes the chemical production of CLA compositions from seed oils with a total CLA content of at least 50%, and with less than 1% contaminating octadecadienoic acid isomers. Another approach, described in WO 97/18320 to Loders Croklaan B. V. uses lipases to selectively esterify 10,12-CLA and thus enrich the 9,11-CLA fraction. The above-described methods, however, do not typically allow for the production of high purity, single isomer CLA, and if single isomer production is achieved on a large scale level, such a process is expected to be expensive.
One method of overcoming the shortcomings of chemical transformation is a whole cell transformation or an enzymatic transformation of linoleic acid, linolenic acid or their derivatives to CLA. It is well known that a biological system can be an effective alternative to chemical synthesis in producing a desired chemical compound where such a biological system is available. The existence of linoleate isomerase enzyme to convert linoleic acid to CLA has been known for over thirty years, however, no one has yet successfully isolated the enzyme. And because it has not yet been isolated, the linoleate isomerase enzyme has not been sequenced.
In many microorganisms, the linoleate isomerase enzyme converts linoleic acid to CLA as an intermediate in the biohydrogenation step. Kepler and Tove have identified this enzyme in Butyrivibrio fibrisolvens (Kepler and Tove, J. Biol. Chem., 1966, 241, 1350). However, they could not solubilize the enzyme; i.e., they were unable to isolate the enzyme in any significantly pure form (Kepler and Tove, J. Biol. Chem., 1967, 242, 5686). In addition, earlier studies have indicated that only compounds which possess a free carboxyl group and a cis-9,cis-12 double bond moieties are isomerized by linoleate isomerase. See Kepler and Tove, Methods in Enzymology, 1969, 14, 105-109, and Kepler et al., J. Biol. Chem., 1970, 245, 3612.
Another research group, Park and colleagues, published an article in J. Food Science Nutrition (Vol. 1: 244-251, 1996), describing the purification of a protein which Park et al. believed to be the Butyrivibrio fibrisolvens linoleate isomerase. However, based on the initial characterization of the enzyme""s activity by Kepler and Tove (see above) and the present inventors"" purification, sequencing and characterization of three demonstrated linoleate isomerases, the present inventors believe that it is very unlikely that the protein that was purified and described by Park et al. is actually a linoleate isomerase. More particularly, it is well established in the art that for successful purification of particulate enzymes, such enzymes must first be converted into a soluble form. Although Park et al. demonstrate that the Butyrivibrio fibrisolvens linoleic acid isomerase is membrane bound, Park et al. describe no such solubilization of the enzyme. Instead, an isolated protein pellet was simply resuspended in phosphate buffer, a procedure that will generally not solubilize any membrane protein, and therefore raises significant doubts about the described purification, particularly in view of previously described purification attempts by Kepler and Tove (J. Biol. Chem. 242:5686-5692, 1967). Indeed, as discussed above, Kepler and Tove had described their extensive but unsuccessful efforts using well accepted solubilization methods (e.g., chelators, organic solvents, high salt, detergents) to attempt to solubilize the isomerase. Furthermore, in contrast to the 19 kD molecular weight of the putative isomerase that was eventually reported by Park et al., the main isomerase activity eluted quite early from the column during purification, indicating an apparent molecular weight of several hundred kD, and not 19 kD. When this initial material was applied to a phenyl sepharose 4B column, multiple broad peaks of activity were observed. This is not typical, and again indicates that the isomerase preparation was heterogeneous, had not been solubilized properly, and was undoubtedly associated with other membrane proteins. One of these activity peaks was then applied to a Superose 6 gel filtration column, yielding a single 19 kD band on gel electrophoresis. Finally, this sample was assayed by Park et al. for isomerase activity using HPLC, which is not appropriate for detection of CLA, since it does not resolve the various positional isomers. The retention time shown for the standard CLA was significantly different than the retention time for the putative CLA formed from linoleic acid using the 19 kD putative linoleate isomerase, and should have lead Park et al. to the firm conclusion that the peak was not CLA, but something else. Therefore, the present inventors believe that the data presented by Park et al. does not support the conclusion that a linoleate isomerase had been purified.
Therefore, there remains a need for purifying and identifying a linoleate isomerase enzyme and/or producing one by recombinant techniques. There also remains a need for finding and identifying an linoleate isomerase enzyme which does not require presence of a free carboxylic acid group in the fatty acid for isomerization. In addition, there remains a need for a method for producing CLA utilizing whole cells or isolated linoleate isomerase enzyme.
The present invention generally relates to isolated linoleate isomerase nucleic acid molecules, isolated linoleate isomerase proteins, immobilized bacterial cells having a genetic modification that increases the action of linoleate isomerase, and methods of using such nucleic acid molecules, proteins and cells to produce CLA.
One embodiment of the invention relates to an isolated 10,12-linoleate isomerase. Included in the invention are linoleate isomerases from Propionibacterium, and particularly, from Propionibacterium acnes, Propionibacterium acidipropionici, and Propionibacterium freudenreichii. Particularly preferred linoleate isomerases include linoleate isomerases from Propionibacterium acnes. In one embodiment, an isolated linoleate isomerase of the present invention converts linoleic acid and linolenic acid to CLA, including (trans,cis)-10,12-linoleic acid. In one embodiment, the protein has a specific linoleic acid isomerization activity of at least about 10 moles CLA mgxe2x88x921 minxe2x88x921.
One embodiment of the present invention relates to an isolated protein, comprising an amino acid sequence selected from the group of: (a) an amino acid sequence selected from the group of SEQ ID NO:42 and SEQ ID NO:61; and, (b) a homologue of the amino acid sequence of (a), wherein the homologue is at least about 35% identical to SEQ ID NO:61 over at least about 170 contiguous amino acids of SEQ ID NO:61. In this embodiment, the protein 10,12-linoleate isomerase enzymatic activity. In one embodiment, the protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence that hybridizes under low, moderate, or high stringency hybridization conditions to the complement of SEQ ID NO:60. In another embodiment, the protein comprises an amino acid sequence comprising at least 15 contiguous amino acids of SEQ ID NO:61, and more preferably, at least 30 contiguous amino acids of SEQ ID NO:61, and even more preferably, at least 45 contiguous amino acids of SEQ ID NO:61. In one embodiment, the protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence comprising at least 24 contiguous nucleotides of SEQ ID NO:60. In a preferred embodiment, the protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:59 and SEQ ID NO:60, with SEQ ID NO:60 being most preferred. In another preferred embodiment, the protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:61, with SEQ ID NO:61 being most preferred. In another embodiment, the protein comprises an amino acid sequence that aligns with SEQ ID NO:73 using Martinez/Needleman-Wunsch DNA alignment method with a minimum match of 9, a gap penalty of 1.10 and a gap length penalty of 0.33, wherein amino acid residues in the amino acid sequence align with at least about 70%, and in another embodiment, with at least about 90%, of non-Xaa residues in SEQ ID NO:73.
In one embodiment, the protein is a soluble enzyme. In another embodiment, the protein comprises a leader sequence which causes insertion of the protein into the membrane of a cell which expresses the protein. In one embodiment, the linoleate isomerase is bound to a solid support, which includes, but is not limited to artificial membranes, organic supports, biopolymer supports and inorganic supports.
Another embodiment of the present invention relates to an isolated antibody that selectively binds to the isolated linoleate isomerase of the present invention.
Yet another embodiment of the present invention relates to a method for producing CLA or derivatives thereof, including contacting an oil, which comprises a compound selected from the group of linoleic acid, linolenic acid, and/or derivatives thereof, with an isolated linoleate isomerase enzyme of the present invention to convert at least a portion of the compound to CLA or derivatives thereof (e.g., when the substrate is a derivative). In one embodiment, the compound is in the form of a triglyceride and the method further includes contacting the oil with a hydrolysis enzyme to convert at least a portion of the triglyceride to free fatty acids. Such a hydrolysis enzyme can include lipases, phospholipases and esterases. The method of the present invention can also include a step of recovering the CLA. The CLA is preferably (trans,cis)-10,12-linoleic acid. The oil can include, but is not limited to, sunflower oil, safflower oil, corn oil, linseed oil, palm oil, rapeseed oil, sardine oil, herring oil, mustard seed oil, peanut oil, sesame oil, perilla oil, cottonseed oil, soybean oil, dehydrated castor oil and walnut oil. In one embodiment of the method, the linoleate isomerase enzyme is bound to a solid support, which can include organic supports, biopolymer supports and inorganic supports.
Another embodiment of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group of: (a) a nucleic acid sequence encoding a protein having 10,12-linoleate isomerase enzymatic activity, wherein the protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:42 and SEQ ID NO:61; (b) a nucleic acid sequence encoding a homologue of a protein of (a), wherein the homologue has 10,12-linoleate isomerase enzymatic activity, and wherein the homologue is at least about 35% identical to SEQ ID NO:61 over at least about 170 contiguous amino acids of SEQ ID NO:61; and/or, (c) a nucleic acid sequence that is fully complementary to any of the nucleic acid sequences of (a) or (b). In one embodiment, the nucleic acid sequence of (b) hybridizes under low, moderate, or high stringency hybridization conditions to the complement of SEQ ID NO:60.
In another embodiment, the homologue comprises at least 15 contiguous amino acids of SEQ ID NO:61, and more preferably, at least 30 contiguous amino acids of SEQ ID NO:61, and even more preferably, at least 45 contiguous amino acids of SEQ ID NO:61. In another embodiment, the nucleic acid sequence of (b) comprises at least 24 contiguous nucleotides of SEQ ID NO:60. The nucleic acid molecule preferably comprises a nucleic acid sequence selected from the group of SEQ ID NO:59 and SEQ ID NO:60, with SEQ ID NO:60 being most preferred. Preferably, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence selected from the group of SEQ ID NO:42 and SEQ ID NO:61, with SEQ ID NO:61 being most preferred.
The isolate nucleic acid molecule of the present invention includes linoleate isomerase nucleic acid molecules from microorganisms including, but not limited to, Propionibacterium, with Propionibacterium acnes, Propionibacterium acidipropionici, and Propionibacterium freudenreichii being particularly preferred. Most preferred linoleate isomerase nucleic acid molecules are from Propionibacterium acnes. 
Also included in the present invention are recombinant molecules, recombinant viruses and recombinant cells which include an isolated nucleic acid molecule of the present invention. In one embodiment, as recombinant cell of the present invention is from a microorganism which includes, but is not limited to, Propionibacterium acnes, Propionibacterium freudenreichii, Propionibacterium acidipropionici, Escherichia coli, Bacillus subtilis, or Bacillus licheniformis, with Escherichia coli, Bacillus subtilis and Bacillus licheniformis being most preferred.
Yet another embodiment of the present invention relates to a method to produce linoleate isomerase, comprising culturing a recombinant cell transfected with an isolated nucleic acid molecule encoding linoleate isomerase.
Another embodiment of the present invention relates to a method for producing CLA or derivatives thereof, including contacting an oil which comprises a compound selected from the group of linoleic acid, linolenic acid, and/or derivatives thereof, with an isolated linoleate isomerase enzyme encoded by the isolated nucleic acid molecule of the present invention to convert at least a portion of the compound to CLA and/or a derivative thereof.
Yet another embodiment of the present invention relates to an immobilized cell having a genetic modification that increases the action of linoleate isomerase. The cell can be any cell, including immobilized bacterial, fungal (e.g., yeast), microalgal, insect, plant or mammalian cells. In one embodiment, the cell is a microorganism which includes, but is not limited to Propionibacterium, Escherichia, Bacillus or yeast cells. In one embodiment, the genetic modification results in overexpression of linoleate isomerase by the cell. The genetic modification can result in at least one amino acid modification selected from the group consisting of deletion, insertion, inversion, substitution and derivatization of at least one amino acid residue of the linoleate isomerase, wherein such modification results in increased linoleate isomerase action, reduced substrate inhibition, and/or reduced product inhibition. In another embodiment, the genetic modification includes transfection of the cell with a recombinant nucleic acid molecule encoding a linoleate isomerase of the present invention, wherein the recombinant nucleic acid molecule is operatively linked to a transcription control sequence. The recombinant nucleic acid molecule can include any of the isolated nucleic acid molecules described above, including a nucleic acid sequence encoding a homologue of linoleate isomerase.
In one embodiment, the recombinant nucleic acid molecule is integrated into the genome of the cell. In another embodiment, the recombinant nucleic acid molecule is a plasmid transformed/transfected into a cell. In another embodiment, the recombinant nucleic acid molecule encoding linoleate isomerase comprises a genetic modification which increases the action of the linoleate isomerase and in another embodiment, the genetic modification reduces substrate and/or product inhibition of the linoleate isomerase.
In another embodiment, an immobilized cell of the present invention can be lysed. The cell can be immobilized by crosslinking with a bifunctional or multifunctional crosslinking agent, including, but not limited to glutaraldehyde.
Yet another embodiment of the present invention relates to a method for producing CLA or a derivative thereof, including contacting an oil which includes a fatty acid selected from the group of linoleic acid, linolenic acid, and derivatives thereof with an immobilized cell having a linoleate isomerase, to convert at least a portion of the compound to CLA or a derivative thereof. Such cells are described above. The cell can be a naturally occurring bacterial cell having a linoleate isomerase, or a genetically modified cell, such as a genetically modified microorganism, as described above. Preferably, a genetically modified cell has increased linoleate isomerase action. The fatty acid can include fatty acids in the form of a triglyceride such that at least a portion of the triglycerides are converted to free fatty acids. Other features of the method are as described above in the method to produce CLA.
Another embodiment of the present invention relates to an isolated lipase-like protein. Such a protein comprises an amino acid sequence selected from the group of: (a) SEQ ID NO:64; and, (b) a homologue of SEQ ID NO:64, wherein the homologue is at least about 35% identical to SEQ ID NO:64. In one embodiment, the protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence that hybridizes under moderate or high stringency conditions to the complement of SEQ ID NO:63. In another embodiment, the protein is encoded by a nucleic sequence comprising at least 24 contiguous nucleotides of SEQ ID NO:63, and more preferably, the protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence represented by SEQ ID NO:63. In one embodiment, the protein comprises amino acid sequence SEQ ID NO:64. In another embodiment, the protein comprises an amino acid sequence having an esterase/lipase/thioresterase active site denoted by ProfileScan Profile No. PS50187. In yet another embodiment, the protein comprises an amino acid sequence having a carboxylesterase type-B active site denoted by ProfileScan Profile No. GC0265. Preferably, the protein has lipase enzymatic activity. Also included in the present invention is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding any of the above-described lipase-like proteins.
Yet another embodiment of the present invention relates to an isolated acetyltransferase-like protein. Such a protein comprises an amino acid sequence selected from the group of: (a) SEQ ID NO:69; and, (b) a homologue of SEQ ID NO:69, wherein the homologue is at least about 40% identical to SEQ ID NO:69 over at least about 60 contiguous amino acid residues of SEQ ID NO:69. In one embodiment, such a protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence that hybridizes under moderate or high stringency conditions to the complement of SEQ ID NO:68. In another embodiment, the protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence represented by SEQ ID NO:68. In one embodiment, the protein comprises amino acid sequence SEQ ID NO:69. In another embodiment, the protein comprises an amino acid sequence having an acetyltransferase (GNAT) family profile denoted by ProScan Profile No. PF00583. Preferably, the protein has acetyltransferase enzymatic activity. Also included in the present invention is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding any of the above-identified acetyltransferase proteins.